The objectives of this grant are to continue our studies on the control of the expression of MHC class I antigens in the placenta of the rat and to explore their role in the outcome of pregnancy using molecular, cytochemical, ultrastructural and embryo transfer techniques. The specific aims are threefold: First, oligonucleotide probes specific for the Pa and Aa genes, based on unique differences between them, will be prepared, and they will be tested using the appropriate inbred and recombinant strains of rats. Then the molecular analysis of Pa+ and Pa- strains will focus on the differences by RFLP analysis in the Pa gene in both types of strains and whether the Pa gene is transcribed in Pa- strains. Second, the three types of control of MHC antigen expression in the placenta will be explored. Genomic imprinting of the Pa and Aa genes and of the genes controlling the expression of the classical (A) and nonclassical (E) class I antigens will be determined by studying their transcription. Constitutive suppression of class I antigens in the labyrinthine trophoblast and of all class II antigens in the placenta will be studied by determining the effect of 5- azacytidine on their expression and on their levels of methylation. Both in vivo experiments and a unique tissue culture system will be used. The effects of seminal fluid and of the timing of embryo transfer will be used to examine the inducible suppression of the classical class I antigens in the outcome of pregnancy will be explored using the transfer of embryos fertilized in vivo or in vitro, different combinations of strains and multiple embryo transfers. The latter approach will provide a unique animal model for exploring the potential role of immunological mechanisms in secondary recurrent spontaneous abortions. The questions addressed have been raised by cogent problems in basic developmental biology and in clinical medicine. The work forms an integral part of our overall hypothesis about the critical role of MHC and MHC- linked genes in reproduction, growth and development, and susceptibility to cancer. A substantial body of evidence from our experimental studies in rats and from clinical studies by us and by others supports this hypothesis. In addition, the work will give some unique insights into the process of in vitro fertilization and into some potential causes for its failure.